Mouse monoclonal antibodies (MAb) were constructed against tumor-associated antigens (TAA) obtained from the spent medium of the M14 human melanoma cell line that had been grown continuously in serum-free medium. Two MAb, D11 and F11, were used to localize TAA in fresh tumor specimens and paraffin-embedded sections. The D11 MAb reacted with a greater incidence against fresh tissue (73%) than with paraffin sections (25%), whereas the F11 MAb reacted with equal incidence against fresh tumor cells and paraffin-embedded sections. Both the D11 and F11 MAb were melanoma-carcinoma associated, and both gave trace reactions with normal colon, liver, and pancreatic tissues. The D11 and F11 MAb either recognize different epitopes of the same antigen or they recognize two distinct TAA. By a direct and a competitive inhibition enzyme-linked immunosorbent assay, the D11 and F11 MAb detected a higher incidence of TAA in the sera of cancer patients than in normal sera. (2)